Methods for increasing gene-editing frequencies in maize cells

Patent metadata

Field	Value
Publication number	US-11718845-B2
Application number	US-202117449011-A
Country	US
Kind code	B2
Filing date	Sep 27, 2021
Priority date	Feb 28, 2017
Publication date	Aug 8, 2023
Grant date	Aug 8, 2023

Abstract

Plant cell cultures as well as related methods, systems, and compositions for increasing the frequency and efficiency of plant genome editing are provided. Various plant cell growth conditions and/or treatments where such increases in gene editing frequencies are obtained are disclosed.

First claim

What is claimed is:

1. A method for making a maize plant protoplast having a genomic modification comprising:
(a) providing genome editing molecules to a maize plant protoplast lacking a cell wall wherein the genome editing molecules are designed for homology-directed repair of a target gene in the plant cell's genome and comprise: (i) an RNA-guided nuclease and a guide RNA and a donor template polynucleotide; (ii) a sequence-specific endonuclease and a donor template polynucleotide; (iii) one or more polynucleotides encoding an RNA-guided nuclease and a guide RNA and a donor template polynucleotide; (iv) a polynucleotide encoding a sequence-specific endonuclease and a donor template polynucleotide; or (v) any combination thereof,
(b) exposing the maize plant protoplast from step (a) to plant cell culture medium comprising a reactive oxygen species (ROS) concentration lowering agent at a concentration of 0.1 millimolar to 10 millimolar and Ca2+ and/or Mg2+ at a concentration of about 40 mM to 150 mM for at least one hour to effect homology-directed repair of the target gene in the plant cell's genome at a frequency that is increased by at least 2-fold in comparison to a control method wherein a control plant cell is provided with the genome editing molecules but is not subjected to a ROS concentration lowering agent at a concentration of 0.1 millimolar to 10 millimolar, wherein the ROS concentration lowering agent is a ROS scavenging agent or an antioxidant; and,
(c) isolating, selecting, identifying, and/or propagating a maize plant protoplast comprising the genome modification, thereby making the maize plant protoplast having a genomic modification.

2. The method of claim 1 , wherein the ROS concentration lowering agent is one or more low-molecular-weight thiol compound(s) wherein the molecular weight of the thiol compound is less than 1,000.

3. The method of claim 2 , wherein the thiol compound(s) are glutathione, cysteine, cysteinyl glycine, gamma-glutamyl cysteine, N-acetylcysteine, cysteine, thiocysteine, homocysteine, lipoic acid, dithiothreitol, or a combination thereof.

4. The method of claim 2 , wherein the thiol compound is gluthathione.

Assignees

• Inari Agriculture Tech Inc

Inventors

• Clement Erik William
• Niu Yajie
• Skraly Frank Anthony

Classifications

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• C12N5/0025

  Culture media for plant cell or plant tissue culture · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title